Doctor of Philosophy / Department of Industrial & Manufacturing Systems Engineering / Jessica L. Heier Stamm / This dissertation focuses on logistics challenges arising in the biofuels industry. Studies have found that logistics costs in the biomass-to-biofuel supply chain (BBSC) account for 35%-65% of total biofuel production cost. This is mainly due to the low density of biomass that results in high costs associated with biomass transportation, storage, and handling in the biomass-to-biofuel supply chain. Densification provides an as-yet-unexplored opportunity to reduce logistic costs associated with biomass-to-biofuel supply chains. This research advances understanding about biomass-to-biofuel supply chain management through new optimization models. As a first step, the author presents an extensive overview of densification techniques and BBSC optimization models that account for biomass densification. This literature review helps the author to recognize the gaps and future research areas in BBSC studies. These gaps direct the author toward the remaining components of the dissertation. In particular, the literature review highlights two research gaps. First, the review indicates that mobile pelleting holds promise for improved BBSC management, but that there is no mathematical optimization model that addresses this opportunity. Second, currently, there does not exist a model that explicitly accounts for farmers’ objectives and their probability to sell biomass to the bioenergy plant in BBSC optimization. To fill the first gap, the author focuses on managing the BBSC considering mobile densification units to account for chances to minimize logistics costs. A mixed integer linear programming model is proposed to manage the BBSC with different types and forms of biomass feedstock and mobile densification units. Sensitivity analysis and scenario analysis are presented to quantify conditions that make mobile densification an attractive choice. The author conducts a case study to demonstrate model applicability and type of analysis that can be drawn from this type of models. The result indicates that mobile pelleting is not an attractive choice under the current economic status. However, modest changes in pelleting cost, satellite storage location fixed cost, and/or travel distances are enough to make mobile pelleting an attractive choice. To fill the second gap, the author introduces a model that explicitly accounts for mobile densification and farmers’ probability to supply a bioenergy plant with biomass feedstock. Farmers’ probability to provide biomass to the bioenergy plant depends on contract attributes, including expected net return and services provided by the bioenergy plant. The proposed model helps the bioenergy plant to meet biofuel demand while considering farmers’ choices that satisfy their own objectives and preferences. The model makes it possible to determine most important factors that influence type of contract offered to each supplier and optimal BBSC design. A case study based on the state of Kansas is conducted to demonstrate how bioenergy plant can benefit from this type of model.

Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.

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In 2023, approximately 2.36 million metric tons of olive oil were produced globally. Olive pomace, a byproduct of the flesh and pits left after olive oil extraction, presents environmental challenges when used as landfill due to its high polyphenol and organic contents, or when combusted due to greenhouse gas emissions. Its potential as animal feed is limited, yet it holds promise for methane production via anaerobic digestion (AD), providing a source of renewable energy. However, the highly crystallized polysaccharides in olive pomace, such as cellulose, hemicellulose, and pectin, impede its conversion to methane, and the high polyphenol content inhibits methanogen growth. To address this, phenolics were extracted from olive pomace, producing a phenolics-extracted olive pomace (PEOP) and a phenolics-rich olive liquid. After further resin-based extraction of phenolics-rich olive liquid, approximately two-thirds of the phenolics were removed, yielding phenolics-extracted olive liquid (PEOL). Enzymatic hydrolysis was conducted on several olive byproduct streams: olive pomace with water, PEOP with PEOL, and PEOP with water, to convert insoluble polysaccharides into reducing sugars that are more readily utilized by methane-producing microorganisms. Various enzymes, including cellulase, hemicellulase, xylanase, and pectinase, were individually treated with olive pomace to determine the optimal hydrolysis time and enzyme concentrations. Response surface methodology (RSM) identified the optimal enzyme cocktail ratio (1.1% cellulase, db, and 0.9% pectinase, db) for achieving the highest reducing sugar contents (22.3 mg/mL), which had 79.1% increase when compared to the control sample (12.5 mg/mL). After 19 days of anaerobic digestion at 37 °C, olive samples before phenolics extraction (olive pomace with water) and olive samples after phenolics extraction (phenolics-extracted olive pomace with phenolics-extracted olive liquid), produced similar amounts of methane (~175 mL CH4/g VS). This indicated that in our experimental settings, the phenolics reduction did not significantly impact methane yields. Carbohydrate profiles may also influence biofuel yields, as hexoses (C6 sugars) are preferred over pentoses (C5 sugars) for end-product production during biotechnical conversion. To explore the effect of carbohydrate profiles on methane production from olive byproducts, two response surface methodology (RSM) coded enzyme cocktail-treated samples with different carbohydrate profiles underwent anaerobic digestion for 19 days at 37 °C, yielding similar amounts of methane (~156 mL CH4/g VS), comparable to the control sample. This suggested that anaerobic digestion can utilize different hexoses and pentoses at similar rates. These findings demonstrated that olive pomace can be used for biomethane production instead of being landfilled or combusted. While enzymatic hydrolysis increased reducing sugar contents, it did not enhance methane yields. Reducing phenolic contents of 2/3 did not improve biomethane yield, and the impact of greater reduction requires further assessment.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25397号 / エネ博第476号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 河本 晴雄 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Increased demand of petroleum, declining fossil fuel reserves, geopolitical instability and the environmentally detrimental effects of fossil fuels have stimulated research to search for alternative sources of energy such as plant derived biofuels. The main feedstocks for production of first generation biofuels (bioethanol) are currently sucrose and starch, produced by crops such as sugarcane, sugarbeet, maize, and cassava. The use of food crop carbohydrates to produce biofuels is viewed as competing for limited agronomic resources and jeopardizing food security. Plants are also capable of storing sugars in their cell walls in the form of polysaccharides such as cellulose, hemicelluloses and pectin, however those are usually cross-linked with lignin, making their fermentation problematic, and are consequently referred to as lignocellulosics. Current technologies are not sufficient to degrade these cell wall sugars without large energy inputs, therefore making lignocellulosic biomass commercially unviable as a source of sugars for biofuel production. In the present study genes encoding for enzymes for cellulosic, hemicellulosic and starch-like polysaccharides biosynthesis were heterologously expressed to increase the amount of fermentable sugars in sugarcane. Transgenic lines heterologously expressing CsCesA, encoding a cellulose synthase from the marine invertebrate Ciona savignyi showed significant increases in their total cellulose synthase enzyme activity as well as the total cellulose content in internodal tissues. Elevation in cellulose contents was accompanied by a rise in hemicellulosic glucose content and uronic acid amounts, while total lignin was reduced in internodal tissues. Enzymatic saccharification of untreated lignocellulosic biomass of transgenic sugarcane lines had improved glucose release when exposed to cellulose hydrolyzing enzymes. Calli derived from transgenic sugarcane lines ectopically expressing galactomannan biosynthetic sequences ManS and GMGT from the cluster bean (Cyamopsis tetragonoloba) were observed to be capable of producing a galactomannan polysaccharide. However, after regeneration, transgenic sugarcane plants derived from those calli were unable to produce the polymer although the inserted genes were transcribed at the mRNA level. While the ectopic expression of Deinococcus radiodurans amylosucrase protein in the cytosol had a detrimental effect on the growth of transgenic lines (plants showed stunted growth through the 18 months growth period in greenhouse), contrastingly targeting the amylosucrase protein into the vacuole resulted in 3 months old transgenic lines which were having high maltooligosaccharide and soluble sugar (sucrose, glucose and fructose) levels in leaves. After 18 months growing in the greenhouse, the mature transgenic lines were morphologically similar to the untransformed lines and also contained comparable maltooligosaccharide and soluble sugar and starch amounts. The non-biosynthesis of galactomannan and amylose polysaccharides in the matured transgenic plants may be due to post-transcriptional protein processing and or protein instability, possibly explainable by other epigenetic mechanisms taking place to regulate gene expression in the at least allo-octaploid species of sugarcane under investigation in this study.

Cellulose from energy crops or agriculture residues can be utilized as a sustainable energy resource to produce biofuels such as ethanol. The process of converting cellulose into solvents and biofuels requires the saccharification of cellulose into soluble, fermentable sugars. However, challenges to cellulosic biofuel production include increasing the activity of cellulose-degrading enzymes (cellulases) and increasing solvent (ethanol) yield while minimizing the co-production of organic acids. This work applies novel surface analysis techniques and fermentation reactor perturbations to quantify, manipulate, and model enzymatic and metabolic processes critical to the efficient production of cellulosic biofuels. Surface analysis techniques utilizing cellulose thin film as the model substrate are developed to quantify the kinetics of cellulose degradation by cellulase as well as the interactions with cellulase at the interfacial level. Quartz Crystal Microbalance with Dissipation (QCM-D) is utilized to monitor the change in mass of model cellulose thin films cast. The time-dependent frequency response of the QCM simultaneously measures both enzyme adsorption and hydrolysis of the cellulose thin film by fungal cellulases, in which a significant reduction in the extent of hydrolysis can be observed with increasing cellobiose concentrations. A mechanistic enzyme reaction scheme is successfully applied to the QCM frequency response for the first time, describing adsorption/desorption and hydrolysis events of the enzyme, inhibitor, and enzyme/inhibitor complexes. The effect of fungal cellulase concentration on hydrolysis is tested using the QCM frequency response of cellulose thin films. Atomic Force Microscopy (AFM) is also applied for the first time to the whole cell cellulases of the bacterium C. thermocellum, where the effect of temperature on hydrolysis activity is quantified. Fermentation of soluble sugars to desirable products requires the optimization of product yield and selectivity of the cellulolytic bacterium, Clostridium thermocellum. Metabolic tools to map the phenotype toward desirable solvent production are developed through environmental perturbation. A significant change in product selectivity toward ethanol production is achieved with exogenous hydrogen and the addition of hydrogenase inhibitors (e.g. methyl viologen). These results demonstrate compensatory product formation in which the shift in metabolic activity can be achieved through environmental perturbation without permanent change in the organism’s genome.

Consolidated bioprocessing (CBP) provides a cost effective cellulose processing strategy, in which enzyme production, substrate hydrolysis, and fermentation of sugars to ethanol are all carried out in a single step by microorganisms. For industrial-scale bioethanol production, CBP-enabling microbes must be able to both efficiently degrade lignocellulosic material to fermentable sugars and synthesize bioethanol with high yields. Microbes with these properties have so far not been identified. Developing naturally occurring cellulolytic isolates with CBP-relevant properties requires a comprehensive understanding of their lignocellulosic hydrolysis mechanism and metabolism. In my quest to find a suitable organism for potential use in CBP, I took to investigate the under-characterized anaerobic bacterium, Clostridium termitidis strain CT1112. C. termitidis produces fermentative hydrogen and ethanol from a variety of lignocellulose derived substrates. I sought to investigate the metabolism of C. termitidis on different substrates and the mechanisms of substrate hydrolysis using a combination of microscopy, comparative bioinformatics, and ‘Omic (transcriptomic and proteomic) analyses. Comparative bioinformatics analyses revealed higher numbers of genes encoding carbohydrate active enzymes (CAZymes) with the potential to hydrolyze a wide-range of carbohydrates, and ‘Omic analyses were used to quantify the levels of expression of CAZymes, including endoglucanases, exoglucanases, hemicellulases and cellulosomal components. While cellulases and cellulosome components were highly expressed on cellulose, xylanases and glucosidases were predominantly expressed on pentoses, and chitinases (as well as cellobiose phosphorylases) were significantly up-regulated on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also observed. The ability to metabolize both hexose and pentose sugars is a highly desirable feature of CBP-relevant organisms. Metabolic profiles in association with ‘Omics analyses showed that hexoses and pentoses are consumed via the Embden-Meyerhof-Parnas and Pentose-Phosphate pathways, respectively, and that the genome content and expression profiles dictate end-product synthesis patterns. Genes and gene-products of enzymes in central metabolism and end-product synthesis were detected in high abundance under all substrate conditions, regardless of the amounts of end-products synthesized. The capabilities described thus far, identifies C. termitidis as a strain of interest for CBP. Further studies are, however, required for its development in to an industry-ready strain for biofuel production. / February 2016

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / Microbial lipids derived from oleaginous yeasts are a promising alternative source of edible oils due to the following advantages: no requirement of broad lands; availability of year-round production; and no food versus fuels controversy. Oleaginous yeast has an inherent ability to accumulate lipids inside cells and their lipids are preferable as starting materials in oleo-chemical industries because of their distinct fatty acid composition. Lignocellulosic biomass is a promising substrate to supply carbon sources for oleaginous yeast to produce lipids due to the high content of polysaccharides and their abundancy. Lignocellulosic-based sugar streams, which can be generated via pretreatment and enzymatic hydrolysis, contained diverse monosaccharides and inhibitors. The major objectives of this study were: 1) to develop a novel purification method to generate clean sugar stream using sorghum stalks after acid pretreatment; 2) to optimize fermentation conditions for Trichosporon oleaginosus to achieve high yields and productivity of microbial lipids using lignocellulosic hydrolysates; 3) to investigate the potentials of sorghum stalks and switchgrass as feedstocks for microbial lipid production using oleaginous yeast strains, such as T. oleaginosus, Lipomyces starkeyi, and Cryptococcus albidus; 4) to develop an integrated process of corn bran based-microbial lipids production using T. oleaginosus; and 5) to develop bioconversion process for high yields of lipids from switchgrass using engineered Escherichia coli. In our investigation, major inhibitory compounds of lignocellulosic hydrolysates induced by pretreatment were acetic acid, formic acid, hydroxymethyl furfural (HMF) and furfural. The activated charcoal was effective in removing hydrophobic compounds from sorghum stalk hydrolysates. Resin mixtures containing cationic exchangers and anionic exchangers in 7:3 ratio at pH 2.7 completely removed HMF, acetic acid, and formic acid from sorghum stalk hydrolysates. T. oleaginosus was a robust yeast strain for lipid production. In the nitrogen-limited synthetic media, total 22 g/L of lipid titers were achieved by T. oleaginosus with a lipid content of 76% (w/w). In addition, T. oleaginosus efficiently produced microbial lipids from lignocellulosic biomass hydrolysates. The highest lipid titers of 13 g/L lipids were achieved by T. oleaginosus using sorghum stalk hydrolysates with a lipid content of 60% (w/w). L. starkeyi and C. albidus also successfully produced microbial lipids using lignocellulosic hydrolysate with a lipid content of 40% (w/w). Furthermore, corn bran was a promising feedstock for microbial lipid production. The highest sugar yields of 0.53 g/g were achieved from corn bran at the pretreatment condition of 1% acid and 5% solid loading. Microbial lipids were successfully produced from corn bran hydrolysates by T. oleaginosus with lipid yields of 216 mg/g. Engineered E. coli also effectively produced lipids using switchgrass as feedstocks. E. coli ML103 pXZ18Z produced a total of 3.3 g/L free fatty acids with a yield of 0.23 g/g. The overall yield of free fatty acids was 0.12 g/g of raw switchgrass and it was 51 % of the maximum theoretical yield. This study provided useful strategies for the development of sustainable bioconversion processes for microbial lipids from renewable biomass and demonstrated the economic viability of a lignocellulosic based-biorefinery.